In one aspect, the present invention relates to the use of bioactive compositions produced, isolated, extracted, based on or derived from either an Aloe vera plant or a portion thereof, for skin enhancement and for pain relief. The invention also relates to immunostimulatory compositions, stabilized immunostimulatory compositions, and methods of making and using such compositions.
The genus Aloe (Liliaceae) is a shrubby tropical/subtropical plant which has succulent and elongated leaves. Of the more than 360 Aloe species known, Aloe barbadensis Miller (Aloe vera Linne) is the most widely used, both commercially and for its therapeutic properties. Aloe vera plants contain two major juice materials: first, a yellow exudate containing a high concentration of anthraquinone compounds that has been used throughout the centuries as a cathartic and for medicinal purges; and second, a clear mucilaginous gel that has been used since ancient times to treat burns and other wounds where it is thought to increase the rate of healing and reduce the risk of infection. See Grindlay, D. Reynolds, T., J. Ethnopharmcol. 1986, 16(2-3), 117-151; and Joshi, S. P., J. Med. Aromat. Plant Sci. 1998, 20(3), 768-773.
For centuries, the Aloe plant has been considered to have, and has been used, for its medicinal and therapeutic properties without any clear understanding or scientific analysis of the bases for such properties. Aristotle was aware that the healing properties of Aloe would be invaluable to soldiers wounded in battle and advised his student Alexander III (“the Great”) to conquer all lands that grew it, especially the island of Socotra off the coast of eastern Africa. Pedanius Dioscorides, a physician in the Roman army, mentioned medicinal Aloes in his encyclopedic Greek herbal De Materia Medica (approximately 75 BC). Further, it is known that the biological activities of fresh Aloe plant decay quite rapidly.
Over that past few decades there has been a high level of interest in developing an understanding of the active components of Aloe that are responsible for its beneficial effects relating to enhanced immune function. The research in this area has focused largely on polysaccharides and other carbohydrate-containing substances such as glycoproteins.
Active chemical substances and mixtures, including for example, polysaccharides or other carbohydrate-containing substances of Aloe leaves have been identified, isolated and stabilized as described in U.S. Pat. Nos. 7,196,072, (“the '072 patent”), 5,902,796, 4,735,935, 4,851,224, 4,917,890, 4,957,907, 4,959,214, and 4,966,892.
One group of the active chemical substances has been referred to as Aloe vera mucilaginous polysaccharides, which are made up of a mixture of polysaccharides. The term “polysaccharides” has been used loosely to include both oligomers and polymers of carbohydrates or simple sugars and is used in a similar manner herein. One group of such polysaccharides has been given the name acemannan.
According to literature, acemannan is an ordered linear polymer of substantially acetylated mannose monomers available from Carrington Laboratories, Inc. (Irving, Tex.), and has been shown in laboratory studies to increase up to 300%, in 48 hours, the replication of fibroblasts in tissue culture, which are known to be responsible for healing burns, ulcers and other wounds of the skin and of the gastrointestinal lining.
Over a three year period, laboratory rats, the stomachs of which react similarly to that of humans, were tested. Acemannan was found to be equivalent to or superior to then current medications used for the treatment of gastric ulcers. Most such products acted to inhibit hydrochloric acid in the stomach. Acemannan worked on a different principle and apparently did not alter the natural flow of digestive acids. See U.S. Pat. No. 5,902,796, column 2, lines 20-25.
Acemannan has been shown to be an inducer of IL-I and prostaglandin E2 (PGE2) production by human peripheral blood adherent cells in culture. IL-I is an important macrophage product reported in the literature to influence the activity and production of lymphocytes, fibroblasts, B-lymphocytes and endothelial cells. See Old, Scientific American, 258(5):59-60, 69-75 (1988).
IL-1 induces fibroblast proliferation which is fundamental to wound healing. IL-1 also: (1) enhances bone marrow activity; it may be therapeutic in individuals whose bone-marrow is depressed; and (2) enhances the immune system in general.
According to literature, a series of experiments with mixed lymphocyte cultures (MLC) has shown that acemannan increases the alloantigenic response of these lymphocytes in a dose-related fashion. Incubation of acemannan with monocytes permitted monocyte-driven signals to enhance the T lymphocyte response to leetin. Related studies on acemannan's effects on MLC have shown an increase in phagocytosis and activity of natural killer cells. In these in vitro test systems, acemannan was allegedly non-toxic and an immunoenhancer.
Acemannan allegedly stimulates lymphocytes to secrete lymphokines and also causes HIV-infected lymphocytes to produce altered glycoproteins (GP-120) by a mechanism similar to that of glucosidase I inhibitors. See Gruters et al., Nature 330:74-77 (1987) and Pal et al., Intervirol. 30:27-35 (1989). Acemannan is phagocytized and apparently pumped to the Golgi/glycoprotein apparatus of the monocyte where it interferes directly with glycoprotein synthesis.
Other uses of Aloe products have been described in, for example, U.S. Pat. Nos. 5,106,616, 5,118,673, 5,308,838, 5,441,943, and 5,443,830. The described studies have primarily focused on the activities of bioactive chemical substances of Aloe vera as antiviral agents, antitumor agents, immunostimulants, immunomodulators, vaccine adjuvants, means of reducing opportunistic infections, means of controlling inflammation, means of stimulating the immune system and wound healing processes.
A number of other products based on polysaccharide-containing fractions isolated from Aloe vera have been published in the patent literature and elsewhere. For example, a polyuronide with a molecular weight between 275,000 and 374,000 daltons is reported to be useful in treatment of surface wounds. (See Farkas, A., U.S. Pat. No. 3,103,466 (1963).)
The 70 kD polysaccharide, Aloeferon, has also been reported to have therapeutic potential. (See Madis, V. H.; Omar, M. M.; Madis, V., U.S. Pat. No. 4,861,761 (1989).)
Other active components isolated from Aloe include a polysaccharide between 420,000 and 520,000 daltons comprised of equal amounts of glucose and mannose. (See Farkas, A., U.S. Pat. No. 3,362,951 (1968).)
In addition, several groups have enzymatically prepared altered polysaccharide compositions from the naturally occurring carbohydrates in Aloe (See Strickland, F. M.; Pelley, R. P.; Kripke, M. L., U.S. Pat. No. 5,824,659 (1998)).
Aloe vera polysaccharides have also been shown in controlled studies to increase the rate of healing in animals. Aloe vera polysaccharides have also been shown to be an effective treatment for gastric ulcers in animal studies.
Anti-inflammatory activity of Aloe vera gel has been reported by both oral testimonies and respected scientific journals. Rubel [Cosmetics and Toiletries, 98:109-114 (1983)] discussed the possible mechanism of the anti-inflammatory effect of Aloe gel. Ukai et al., [Journal of Pharmacobio-Dynamics, 6(12):983-990 (1983)] noted anti-inflammatory activity of polysaccharides extracted from the fruiting bodies of several fungi. The polysaccharides allegedly demonstrated a significant inhibitory effect on carrageenan-induced edema. One of the polymers, O-acetylated-D-mannan (T-2-HN), in addition demonstrated a more marked inhibitory effect than phenylbutazone on scald hyperalgesia. Ukai et al., supra. The assertion in this article, that the polysaccharide is allegedly free from protein and lipids suggests that the anti-inflammatory effect is due to the acetylated mannan only.
Other researchers have also reported anti-inflammatory effects of complex polysaccharides [Saeki et al., Japanese Journal of Pharmacology, 24(1):109-118 (1974)], glycoproteins [Arita et al., Journal of Biochemistry, 76(4):861-869 (1974)] and sulfated polysaccharides [Rocha et al., Biochemical Pharmacology, 18:1285-1295 (1969)].
It is therefore possible that mucilaginous gel, a component of the Aloe vera plant, which is essentially a polysaccharide, and components therein, are responsible in part for Aloe vera's medicinal properties. The controversy over whether the polysaccharide is a glucomannan, mannan, pectin, or of some other composition, is allegedly resolved by a series of chemical purification steps. Yagi et al., [Planta Medica, 31(1):17-20 (1977)], using a slightly modified extraction method; isolated acetylated mannan (Aloe mannan) from Aloe arborescens Miller var. natalensis. Ovodova [Khim, Prior, Soedin, 11(1):325-331 (1975)], however, earlier isolated pectin as the main component of the same Aloe species.
A number of other pharmacology studies have been conducted on Aloe vera gel in recent times. Results have included more rapid healing of radiation burns [Rowe, J. Am. Pharm. Assoc., 29:348-350 (1940)] and accelerated healing of wounds [Lushbaugh et al., Cancer, 6:690-698 (1953)]. Thermal burns treated with Aloe vera gel heal much faster than untreated burns [Ashley et al., Plast. Reconstr. Surg., 20:383-396 (1957), Rovatto, supra, Rodriguez-Bigas et al., J. Plast. Reconstr. Surg., 81:386-389 (1988)]. The gel is useful in treating leg ulcers [El Zawahry et al., Int. J. Dermatol., 12:68-73 (1973)] and in hastening post surgical healing (Payne, Thesis submitted to Faculty of Baylor University, Waco, Tex., MS Degree). Experimental evidence suggests that extracts of Aloe vera have anti-infectious properties [Solar, Arch. Inst. Pasteur Madagascar, 47:9-39 (1979)] and enhance phagocytosis [Stepanova, Fiziol. Akt. Veshchestva, 9:94-97 (1977)].
During commercial processing and quality control of Aloe products, microbiological analysis is recognized as an important issue (Waller T A, et al. In Aloes: The genus Aloe; Reynolds T, Ed.; CRC Press: NY, 2004; Chapter 8, pp 139-205). Microbiological organisms can originate from both exogenous sources such as the environment and from within the Aloe plant itself. The endogenous bacterial flora are microorganisms that naturally exist within Aloe that have evolved to grow well in that unique environment. The primary interest in endogenous bacteria has been with respect to controlling its potential overgrowth that can result with improper post-harvest processing methods (e.g. lack of or incorrect pasteurization). Furthermore, it has been highlighted that high bacterial load in Aloe products is likely to result in loss of biological activity. This has been reported by the observation that high bacterial content (>100,000 organisms/gram of gel) is correlated with loss of Aloe's ability to protect the skin immune system from UVB-induced suppression (Waller T A, et al. In Aloes: The genus Aloe; Reynolds T, Ed.; CRC Press: NY, 2004; Chapter 8, pp 139-205).
References in the art describe bacterial components in Aloe with respect to endotoxin (also referred to as lipo-polysaccharides) contamination as a confounding factor in evaluating the biological activity of purported active substances such acemannan polysaccharide. For example, this point was stressed by Tizard (Tizard I R and Ramamoorthy L. In Aloes: The genus Aloe; Reynolds T, Ed.; CRC Press: NY, 2004; Chapter 13, pp 311-332): “It is difficult to produce Aloe carbohydrate solutions free of contaminating endotoxin. Early studies on this material contained small but significant quantities of bacterial endotoxin and it is possible that some of the biological activities ascribed to acemannan may have been endotoxin effects”. Thus, bacterial products in Aloe preparations are considered contaminants.
U.S. Pat. No. 5,902,796 describes various methods and separation processes for obtaining different factors from Aloe vera. One factor described is called a “microparticulate factor” that can be obtained as a pellet by centrifugation at 20,000 g of the supernatant obtained from a low speed centrifugation (180 g) of an Aloe solution. Methods are also described for obtaining this factor using filtration. This microparticulate factor is an activator of macrophages. The size of this microparticulate factor is estimated to be approximately 1 μm.
The characterization of the microparticles was described in a later paper (Ni Y, et al, International Immunopharmacology, 2004, 4: 1745-1755). The microparticles were reported to contain galactose-rich polysaccharide(s) with the following composition: 40.2% galactose, 32.2% mannose, 20.6% glucose and other minor sugars. This paper identifies the microparticles as degenerated cellular organelles of mesophyll cells, as summarized: “Following disruption of pulp by homogenization, three components were isolated by sequential centrifugation. They were thin clear sheets, microparticles and a viscous liquid gel, which correspond to cell wall, degenerated cellular organelles and liquid content of mesophyll cells based on morphological and chemical analysis”. Clearly these microparticles are not believed to be bacteria but rather have a defined carbohydrate composition and are plant structural components (i.e. degenerated cellular organelles of mesophyll cells).
Gram positive micrococcus bacterial species are the most prevalent Aloe-associated organisms. These bacteria have evolved to grow well within the Aloe plant and do not grow very well in other environments (Waller T A, et al. In Aloes: The genus Aloe; Reynolds T, Ed.; CRC Press: NY, 2004; Chapter 8, pp 139-205).
Recent research indicates that consumption of foods containing certain gram positive bacteria can have a beneficial effect on the immune system. This has been most extensively studied with respect to the ingestion of the probiotic lactic acid bacteria Lactobacillus and Bifidobacterium strains found in yoghurt and similar foods (See, Nutr Rev., 2006, 64: 1-14). Many of these immune enhancing effects do not require viable bacterial cells since they can be mimicked by consumption of heat killed organisms.
Problems often associated with aging include a loss of skin elasticity and muscle or joint pain. These symptoms may or may not be related and while significant steps are being made in the understanding of the aging process, much is still unknown. Treatments for skin elasticity are often cosmetic in nature only. By covering the skin with a cosmetic, a more youthful appearance can be obtained in some instances. Some products actually glue the loose skin making it appear more taught. There are also surgical methods which can involve removing excess skin and/or tightening the skin in procedures commonly referred to as “face lifts”. If, however, skin elasticity could be improved by the use of preparations which are ingested or applied topically, and which actually effect the elasticity of the skin directly, surgery could be postponed or avoided completely and fewer cosmetic products might be needed.
Similarly, there are many possible causes of joint pain including arthritis and many strategies to treat them. These include topical products, pharmaceutical preparations and even joint replacement surgery. Still, as to either joint pain, or skin elasticity, improvements in cure methods are necessary. If a product whose active ingredient is all-natural could be used to provide primary relief, or could be combined with other strategies, the results would be highly desirable.